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The Coomassie Blue G-250 dye is reddish/brown with an absorbance maximum of 465 nm and interacts with proteins in the Bradford method producing a change in color. The blue color produced under acidic conditions is the result of the dye reacting with arginine and to a lesser extent with lysine, histidine, tyrosine, tryptophan, and phenylalanine residues in proteins. The absorbance maximum is 595 nm (the absorption range is between 575 and 615 nm) and 0.2–20 μg of protein can be detected. The method is the easiest and fastest of the protein determination methods currently in use.
Figure 1. Determination of Protein Concentration by the Bradford Coomassie Blue G-250 Method.
1. Spectrophotometer absorbing in the visible range and 1 mL plastic or glass cuvettes.
2. Dissolve 100 mg Coomassie Brilliant Blue G250 in 100 mL of 85 % (v/v) phosphoric acid and 50 mL 95 % (v/v) ethanol. After the dye is dissolved adjust the volume to 1 L with water. The reagent is stable for several months. Should a precipitate form, filter through filter paper and determine the standard curve again.
1. Set the spectrophotometer to read at 595 nm, allowing 15 min for the instrument to equilibrate.
2. Prepare a BSA protein standard at 1 mg/mL concentration in duplicate. Dilute the protein standard in a volume of 20 μL to give five concentrations over a range of 10–50 μg protein. Each dilution of each of the two standards should be done in duplicate to produce four readings per protein concentration.
3. Add 20 μL of protein solution to 1 mL of dye reagent, mix, incubate for 2 min at room temperature, and measure the absorbance in a cuvette.
1. Advantages of the Bradford Coomassie Blue G-250 assay: (a) The method is fast. (b) The method is simple. (c) The reagents are stable for long periods of time. (d) The volume of reagents can be reduced and the assay performed in a 96-well microtiter plate. (e) The method is sensitive and (f) can be automated.
2. Disadvantages of the Bradford Coomassie Blue G-250 assay: (a) The dye stains cuvettes. Cuvettes can be cleaned by washing with a dilute sodium dodecyl sulfate solution. (b) Dye binding depends on the basic amino acid content that can vary between proteins. (c) Concentrated protein solutions can form a precipitate upon contact with the dye reagent. If this is observed, the protein solution should be diluted to determine protein concentration, and (d) the method is destructive to proteins; that is, once the protein sample has reacted with the dye, the protein cannot be used for other assays.
3. It is advisable to dissolve the protein standard in duplicate. If a protein standard is weighed or solubilized incorrectly in a single preparation, then all protein determinations will be inaccurate.
4. Protein determinations with the Coomassie Blue reagents should always be considered as a relative concentration of protein. It is accepted practice to express the relative concentrations of proteins during protein purification steps, provided that all protein concentrations are determined with the same method.
5. The Coomassie Blue protein determination methods all produce a nonlinear curve, whilst absorbance at 280 nm produces a straight line as protein concentration increases.
6. The Coomassie Blue protein curves all proceed from the origin of the graph, provided that the correct "blank" or "control" has been used.
Reference
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